Assessment of genetic diversity within and among germplasm accessions in cultivated sorghum using microsatellite markers

Microsatellite markers are increasingly being used in crop plants to discriminate among genotypes and as tools in marker-assisted selection. Here we evaluated the use of microsatellite markers to quantify the genetic diversity within as well as among accessions sampled from the world germplasm collection of sorghum. Considerable variation was found at the five microsatellite loci analysed, with an average number of alleles per locus equal to 2.4 within accessions and 19.2 in the overall sample of 25 accessions. The collection of sorghum appeared highly structured genetically with about 70% of the total genetic diversity occurring among accessions. However, differentiation among morphologically defined races of sorghum, or among geographic origins, accounted for less than 15% of the total genetic diversity. Our results are in global agreement with those obtained previously with allozyme markers. We were also able to show that microsatellite data are useful in identifying individual accessions with a high relative contribution to the overall allelic diversity of the collection. Received: 10 August 1999 / Accepted: 27 August 1999     

Assessing population genetic structure of sorghum landraces from North-western Morocco using allozyme and microsatellite markers

 The level of genetic diversity and the population genetic structure of sorghum landraces from North-western Morocco have been investigated based on direct field-sampling using both allozyme and microsatellite markers. As expected, microsatellite markers showed a much higher degree of polymorphism than allozymes, but relative measures of genetic structure such as Wright’s inbreeding coefficient F _IS and Nei’s coefficient of genetic differentiation G _ST were similar for the two sets of markers. Substantial inbreeding was found to occur within fields, which confirms that sorghum is predominantly selfing under cultivation. Most of the genetic diversity in Moroccan landraces occurs within fields (more than 85%), as opposed to among fields or among regions, a result which contrasts to those of studies based on accessions from germplasm collections. It is suggested that individual fields of sorghum constitute valuable units of conservation in the context of in situ conservation practices. Received: 8 December 1998 / Accepted: 28 December 1998     

Evaluation of the genetic diversity of Laiwu pigs using twenty-seven microsatellite markers

The Laiwu pig, an indigenous pig breed known for extremely high intramuscular fat content, is a well-preserved ancient breed due to long-term natural and artificial selections. In this study, using 27 microsatellite markers jointly recommended by the International Society of Animal Genetics (ISAG) and the Food and Agriculture Organization (FAO), we investigated the genetic diversity of the Laiwu pig breed. The genetic diversity of Laiwu pigs is dramatically low, with the observed heterozygosity ranging from 0.067 to 0.767. Among the 27 microsatellite markers, 10 were high polymorphic loci, 10 were moderate polymorphic loci, six were low polymorphic loci, and no polymorphism was detected at one locus (IGFI). Further analyses with the 10 high polymorphic loci and five moderate polymorphic loci revealed that the Laiwu pig breed was inbred and heterozygous deficient to some extent, but not severely, and that the Laiwu pigs were relatively pure, with almost no hybridization with other breeds. Two subgroups of the current 13 Laiwu pig pedigrees were identified. These results suggest that the Laiwu pig breed has a low diversity and a conservation program must be developed to preserve the “Laiwu pig” gene pool.     

Genetic characterization and breed assignment in five Italian sheep breeds using microsatellite markers

The knowledge of the genetic relationship and admixture among neighbouring populations is crucial for conservation efforts. The aim of this study was to analyse the genetic diversity of five Italian sheep breeds (Appenninica, Garfagnina Bianca, Massese, Pomarancina and Zerasca) using a panel of 24 microsatellite markers. Blood samples from 226 individuals belonging to the aforementioned populations were obtained and genotyped. All the investigated breeds showed a significant heterozygote deficiency caused by the high level of inbreeding indicated also by the high level of F_IS (0.146). Genetic differentiation between breeds was moderate (F_ST = 0.05) but significant and the individuals could be assigned to their breeds with an high success rate even if the inter-individual distances showed that few animals clustered separately from the other individuals of the same breed, especially for Pomarancina breed. The genetic distances reflect the historical knowledge of these breeds and some patterns of ancestral and recent gene flow between neighbour populations arise. The clustering analysis detects the presence of six clusters. Massese and Zerasca breeds were grouped together as well as Appenninica and Pomarancina with the latter forming two distinct clusters equally represented. The formation of this last breed is occurred with the absorption of individuals of the Appenninica breed and the gene flow probably continued in these recent years allowing the presence of a population substructure for Pomarancina breed. Such substructure supports the high level of heterozygote deficiency found for this breed despite the relatively high population size. The five populations analysed presented some genetic similarities but a clear uniqueness of the populations has been showed for almost all of them. Special attention to monitor genetic variability and to organize mating plans should be given especially for the three endangered breeds.     

Assessment of genetic diversity of Czech sweet cherry cultivars using microsatellite markers

We analyzed 24 sweet and wild cherry genotypes collected in Czech Republic to determine genetic variation, using previously described 16 SSR primers to adapt a fast, reliable method for preliminary screening and comparison of sweet cherry germplasm collections. All SSRs were polymorphic and they were able all together to distinguish unambiguously the genotypes. These SSR primers generated 70 alleles; the number of alleles per primer ranged from 2 to 7, with a mean of 4.4 putative alleles per primer combination. The primer UDP-98-412 gave the highest number of polymorphic bands (totally 7), while Empa2 and Empa3 gave the lowest number (2). The allele frequency varied from 2.1% to 87.5%. We observed 10% of unique alleles at different loci. The observed heterozygosity value ranged from 0.25 to 0.96 with an average of 0.72 while expected heterozygosity value varied from 0.22 to 0.75 with an average of 0.59. The PIC value ranged from 0.21 to 0.71 with a mean value of 0.523. Cluster analysis separated the investigated cultivars in two groups. High level of genetic diversity obtained in the collection and proved to be sufficiently genetically diverse and therefore these genotypes would be useful to breeders for the development of new cherry cultivars.     

Assessing the genetic consequences of flower-harvesting in Rhododendron decorum Franchet (Ericaceae) using microsatellite markers

Rhododendron decorum is widely distributed shrub in southwest China, and its flower is a favorite food of the local people. To investigate the impacts of harvesting, we genotyped 8 nuclear microsatellite loci in a total of 247 individuals from 10 natural populations and 4 flower-harvesting populations. No significant differences in allelic richness, effective number of alleles, private allelic richness, heterozygosity and effective population size were found among the natural and flower-harvesting populations. Differentiation between the 14 populations is relatively low (F_ST = 0.107). R. decorum showed high levels of intra-population genetic diversity. AMOVA analysis indicated that over 89% of the variation was contained within the populations, and that only 0.47% of the variation was attributed by human harvesting practices. Cluster analysis revealed two basic clusters related to the plants' geographical locations. Our results indicate that historical flower-harvesting practices do not lead to loss of genetic variation in R. decorum.     

Comparative analysis of genetic diversity and population genetic structure in Abies chensiensis and Abies fargesii inferred from microsatellite markers

Abies chensiensis Tieghem and Abies fargesii Franchet are two closely related tree species of Pinaceae endemic to China. A. chensiensis is usually found scattered in small forest fragments, whereas A. fargesii is a dominant member of coniferous forest. To evaluate the genetic effect of fragmentation on A. chensiensis, a total of 24 populations were sampled from the whole distribution of the two species. Seven nuclear microsatellite loci were employed to analyze comparatively the genetic diversity and population genetic differentiation. Both A. chensiensis and A. fargesii have high level within-population genetic diversity and low inter-population genetic differentiation. Low microsatellite differentiation (2.1%) between A. fargesii and A. chensiensis was observed. But microsatellite marker was able to discriminate most populations of these two species. Compared to A. fargesii, A. chensiensi has lower allelic diversity and higher genetic differentiation among populations. It suggested the existence of negative genetic impacts of habitat fragmentation on A. chensiensis.     

High-throughput microsatellite markers discovery for the Sichuan Hill Partridge (Arborophila rufipectus) and assessment of genetic diversity in the Laojunshan population

Sichuan Hill Partridge (Arborophila rufipectus) is a globally endangered species endemic to China. However, the genetic diversity of this species was poorly studied due to lack of molecular markers. We used 454 pyrosequencing to discover microsatellites from the A. rufipectus genome in this study. A total of 6280 di-nucleotides, 8139 tri-nucleotides, and 11,987 tetra-nucleotides with sufficient flanking region for primer design were screened with in silico mining. Ultimately, 18 tetra-nucleotide polymorphic loci were first identified and used to examine the genetic diversity in the Laojunshan population. The number of alleles per locus ranged from 5 to 13, observed and expected heterozygosity varied from 0.292 to 0.917 and 0.722 to 0.909, respectively. These results revealed that this endangered species' genetic diversity as not as low as expected. However, a heterozygote deficit was found in the Laojunshan population. We proposed that the management should focus on increasing the connectivity of remnant patches and fragments to increase the opportunity of greater gene flow among fragmented populations of A. rufipectus. This is the first time that polymorphic microsatellite markers were reported for A. rufipectus. These markers provide a valuable resource for future population genetics studies and for the management and conservation of this species.     

Assessing genetic diversity in Ziziphus jujuba ‘Jinsixiaozao’ using morphological and microsatellite (SSR) markers

Ziziphus jujuba ‘Jinsixiaozao’ is one of the most elite Chinese jujube variety with a long cultivation history. There are many different types and newly developed accessions of ‘Jinsixiaozao’ reported, however the names are in chaotic. For the accurate identification of the accessions and estimating the genetic diversity, the diversity and relationships of the 45 ‘Jinsixiaozao’ accessions were evaluated by 49 morphological traits and 24 highly polymorphic genomic SSR primers. The UPGMA dendrogram based on morphological traits separated the accessions into four major groups with Euclidean distance ranging from 4.26 to 12.26. Six of 24 SSR primers produced polymorphic patterns with a total of 17 alleles. Cluster analysis using UPGMA and Jaccard‘s coefficient grouped the accessions into eight groups. The SSR markers failed to distinguish the majority of the analyzed accessions, and a negative percentage of variation was partitioned. These results indicated low genetic diversity among the collected accessions. The mantel test revealed a weak negative correlation (r = −0.051) between the morphological dissimilarity matrix and that based on SSR markers.     

Assessment of the genetic diversity and genetic relationships of Lilium in China using ISSR markers

China is a center of natural distribution and diversity of genus Lilium around the world. In the study, the genetic diversity and genetic relationships of Lilium in China were analyzed by inter-simple sequence Repeat (ISSR) markers. The 6 highly polymorphic ISSR primers were selected to amplify the 20 Lilium species. The results showed that a total of 114 DNA bands were amplified, all of which were polymorphic loci (P = 100%), the effective number of alleles (Ne) was 1.2753, and the difference value between observed number of alleles (Na) and effective number of alleles (Ne) was 0.7247, and the Nei's genetic diversity (H) was 0.2048, and the Shannon's information index (I) was 0.3503. These results indicated that there is significant genetic difference among Lilium species in China. Taking the average genetic similarity coefficient (Gs) 0.5313 as the threshold, the 20 tested Lilium species were clustered into 5 groups, which was not entirely consistent with traditional clustering by morphological traits. The results obtained from this study can provide a reference for the molecular study of Lilium germplasm resources.     

Determination of genetic diversity of wild and cultured topmouth culter (Culter alburnus) inhabiting China using mitochondrial DNA and microsatellites

The topmouth culter (Culter alburnus) is one of the most commercially important freshwater fish species inhabiting China. However, very limited information is available regarding its genetic diversity and population structure, thus hindering the effective management of this fish stock. Understanding the genetic diversity of wild and cultured topmouth culter populations is highly relevant for successful hatchery management. This study evaluated the genetic diversity and structure of five wild and two cultured populations of topmouth culter in China by using microsatellites and mitochondrial DNA. The genetic diversity of wild populations was found to be lower than that of cultured populations. This finding indicates that wild topmouth culter resources should be protected to prevent further degeneration and extinction. Moreover, it demonstrated that cultured populations have greater breeding potential than wild ones. Subdivisions among wild populations were observed, which should be considered as different units for conservation and hatchery management.     

Microsatellite-based molecular diversity of bread wheat germplasm and association mapping of wheat resistance to the Russian wheat aphid

Genetic diversity of a set of 71 wheat accessions, including 53 biotype 2 Russian wheat aphid (RWA2)-resistant landraces and 18 RWA2 susceptible accessions, was assessed by examining molecular variation at multiple microsatellite (SSR) loci. Fifty-one wheat SSR primer pairs were used, 81 SSR loci were determined, and 545 SSR alleles were detected. These SSR loci covered all the three genomes, 21 chromosomes, and at least 41 of the 42 chromosome arms. Diversity values averaged over SSR loci were high with mean number of SSR alleles/locus = 6.7, mean Shannon’s index (H) = 1.291, and mean Nei’s gene diversity (He) = 0.609. The three wheat genomes ranked as A > D > B and the homoeologous groups ranked as 7 > 3  > 1 > 2 > 6 > 5 > 4 based on the number of alleles per locus. Xgwm136 on chromosome arm 1AS is the most polymorphic SSR locus with the largest number of observed and effective alleles and the highest H and He. Among all 2485 pairs of wheat accessions, genetic distance (GD) ranged from 0.054 to 1.933 and averaged 0.9832. A dendrogram based on GD matrix showed that all the wheat accessions could be grouped into distinct clusters. Most of the susceptible cultivars (13/18) were clustered into groups that contains all or mostly susceptible accessions. Most of the U.S. cultivars belong to a group that is distinguishable from all the different RWA2 resistant groups. Diversity analysis was also conducted separately for subgroups containing 53 RWA2-resistant accessions and 18 RWA2-susceptible accessions. Association mapping revealed 28 SSR loci significantly associated with leaf chlorosis, and 8 with leaf rolling. New chromosome regions associated with RWA2 resistance were detected, and indicated existence of new RWA resistance genes located on chromosomes of all other homoeologous groups in addition to the groups 1 and 7 in bread wheat. This information is helpful for development of mapping populations for RWA2 resistance genes from different phylogenetic groups, and for wise utilization of the RWA-resistant germplasm in wheat breeding programs.     

Assessment of genetic diversity in Azadirachta indica using AFLP markers

 Genetic diversity was estimated in 37 neem accessions from different eco-geographic regions of India and four exotic lines from Thailand using AFLP markers. Seven AFLP selective primer combinations generated a total of 422 amplification products. The average number of scorable fragments was 60 per experiment, and a high degree (69.8%) of polymorphism was obtained per assay with values ranging from 58% to 83.8%. Several rare and accession-specific bands were identified which could be effectively used to distinguish the different genotypes. Genetic relationships within the accessions were evaluated by generating a similarity matrix based on the Jaccard index. The phenetic dendrogram generated by UPGMA as well as principal correspondence analysis separated the 37 Indian genotypes from the four Thai lines. The cluster analysis indicated that neem germplasm within India constitutes a broad genetic base with the values of genetic similarity coefficient ranging from 0.74 to 0.93. Also, the Indian genotypes were more dispersed on the principal correspondence plot, indicating a wide genetic base. The four lines from Thailand, on the other hand, formed a narrow genetic base with similarity coefficients ranging from 0.88 to 0.92. The lowest genetic similarity coefficient value (0.47) was observed between an Indian and an exotic genotype. The level of genetic variation detected within the neem accessions with AFLP analysis suggests that it is an efficient marker technology for delineating genetic relationships amongst genotypes and estimating genetic diversity, thereby enabling the formulation of appropriate strategies for conservation and tree improvement programs. Received: 20 October 1998 / Accepted: 28 November 1998     

The use of microsatellite markers for detection of genetic diversity in barley populations

 A barley lambda-phage library was screened with (GA)n and (GT)n probes for developing microsatellite markers. The number of repeats ranged from 2 to 58 for GA and from 2 to 24 for GT. Fifteen selected microsatellite markers were highly polymorphic for barley. These microsatellite markers were used to estimate the genetic diversity among 163 barley genotypes chosen from the collection of the IPK Genebank, Germany. A total of 130 alleles were detected by 15 barley microsatellite markers. The number of alleles per microsatellite marker varied from 5 to 15. On average 8.6 alleles per locus were observed. Except for GMS004 all other barley microsatellite markers showed on average a high value of gene diversity ranging from 0.64 to 0.88. The mean value of gene diversity in the wild forms and landraces was 0.74, and even among the cultivars the gene diversity ranged from 0.30 to 0.86 with a mean of 0.72. No significant differences in polymorphism were detected by the GA and GT microsatellite markers. The estimated genetic distances revealed by the microsatellite markers were, on average , 0.75 for the wild forms, 0.72 for landraces and 0.70 among cultivars. The microsatellite markers were able to distinguish between different barley genotypes. The high degree of polymorphisms of microsatellite markers allows a rapid and efficient identification of barley genotypes. Received: 26 November 1997 / Accepted: 19 January 1998     

Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers

China is rich in chicken genetic resources, and many indigenous breeds can be found throughout the country. Due to poor productive ability, some of them are threatened by the commercial varieties from domestic and foreign breeding companies. In a large-scale investigation into the current status of Chinese poultry genetic resources, 78 indigenous chicken breeds were surveyed and their blood samples collected. The genomes of these chickens were screened using microsatellite analysis. A total of 2740 individuals were genotyped for 27 microsatellite markers on 13 chromosomes. The number of alleles of the 27 markers ranged from 6 to 51 per locus with a mean of 18.74. Heterozygosity (H) values of the 78 chicken breeds were all more than 0.5. The average H value (0.622) and polymorphism information content (PIC, 0.573) of these breeds suggested that the Chinese indigenous chickens possessed more genetic diversity than that reported in many other countries. The fixation coefficients of subpopulations within the total population (F _ST) for the 27 loci varied from 0.065 (LEI0166) to 0.209 (MCW0078), with a mean of 0.106. For all detected microsatellite loci, only one (LEI0194) deviated from Hardy-Weinberg equilibrium (HWE) across all the populations. As genetic drift or non-random mating can occur in small populations, breeds kept on conservation farms such as Langshan chicken generally had lower H values, while those kept on large populations within conservation regions possessed higher polymorphisms. The high genetic diversity in Chinese indigenous breeds is in agreement with great phenotypic variation of these breeds. Using Nei’s genetic distance and the Neighbor-Joining method, the indigenous Chinese chickens were classified into six categories that were generally consistent with their geographic distributions. The molecular information of genetic diversity will play an important role in conservation, supervision, and utilization of the chicken resources.     

Evaluation of genetic diversity in Chinese indigenous chicken breeds using microsatellite markers

China is regarded as one of the domestication cen-ters for chickens and archaeological studies provided evidence of chicken domestication in northern Chinaas early as 6000 BC[1]. At present, China has the larg-est chicken population in the world, represen…     

De novo assembly and characterization of transcriptome using Illumina sequencing and development of twenty five microsatellite markers for an endemic tree Juglans hopeiensis Hu in China

The Chinese walnut (Juglans hopeiensis Hu) is an endemic temperate tree species and narrowly distributed in China. However, there are still few specific molecular markers for understanding genetic diversity of this walnut. In this study, more than 44 million sequencing reads were generated using Illumina sequencing technology. De novo assembly yielded 93,822 unigenes with an average length of 731 bp. Based on sequence similarity search with known proteins, a total of 39,708 (42.3%) genes were identified. Searching against the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) indicated that 15,903 (17.0%) unigenes. To contribute to its conservation and management, twenty five microsatellite markers were identified in J. hopeiensis were screened for polymorphism across 27 Chinese walnut tree individuals from two locations. The number of alleles ranged from two to nine, observed heterozygosity ranged from 0.016 to 0.933 (mean 0.468), and expected heterozygosity from 0.022 to 0.823 (mean 0.462). The polymorphic information content (PIC) ranged from 0.012 to 0.831 (mean 0.437). The development of these new microsatellite markers will be useful for studying population genetic structure, evolutionary ecology, conservation, and genetic breeding of this endemic walnut tree or other Juglans species.     

Genetic diversity of invasive Oreochromis spp. (tilapia) populations in Guangdong province of China using microsatellite markers

Ten microsatellites were used to assess the genetic diversity and genetic differences of six wild Oreochromis spp. populations in the primary rivers of Guangdong Province of China, where natural populations have been established. Significant genetic differentiation was observed in Oreochromis spp. populations using “FST” and exact tests. The six populations can be divided into two groups based on the genetic similarity indices. The phylogenetic tree indicated that the HUI (Huizhou) and the SG (Shaoguan) populations formed one cluster, whereas the H (Huazhou), M (Meihua), MU (Muzhou), and ZQ (Zhaoqing) populations formed another cluster. The average mean levels of the observed heterozygosity in the six populations were 0.3972, 0.4264, 0.6977, 0.6335, 0.6680, and 0.6829, respectively. The high genetic diversity observed in most wild tilapia populations suggests that most wild populations are large in size, which is a finding that is supported by field investigations.